Process for producing N-acetylneuraminic acid

ABSTRACT

The present invention provides a process for economically producing N-acetylneuraminic acid without using expensive materials such as pyruvic acid and phosphoenolpyruvic acid. The process comprises: allowing (i) a culture of a microorganism having N-acetylneuraminic acid aldolase activity or N-acetylneuraminic acid synthetase activity, or a treated matter of the culture, (ii) a culture of a microorganism capable of producing pyruvic acid or a treated matter of the culture, or a culture of a microorganism capable of producing phosphoenolpyruvic acid or a treated matter of the culture, (iii) N-acetylmannosamine, and (iv) an energy source which is necessary for the formation of pyruvic acid or phosphoenolpyruvic acid to be present in an aqueous medium to form and accumulate N-acetylneuraminic acid in the aqueous medium; and recovering N-acetylneuraminic acid from the aqueous medium.

BACKGROUND OF THE INVENTION

The present invention relates to a process for producingN-acetylneuraminic acid by using a microorganism havingN-acetylneuraminic acid aldolase activity or N-acetylneuraminic acidsynthetase activity.

It is known that N-acetylneuraminic acid can be produced by extraction,decomposition or by use of enzymes.

An example of a known method by extraction is extraction from a nest ofsea swallows, etc. [Carbohydrate Research, 56, 423 (1977)].

An example of a known method by decomposition is decomposition ofcolominic acid, which is an N-acetylneuraminic acid polymer produced byEscherichia coli, etc. [J. Biochem., 82, 1425 (1977)].

Known methods utilizing enzymes include the following: methods usingN-acetylneuraminic acid aldolase, pyruvic acid and N-acetylmannosamine[J. Am. Chem. Soc., 110, 6481 (1988); J. Am. Chem. Soc., 110, 7159(1988)]; a method using N-acetylneuraminic acid aldolase, pyruvic acidand N-acetylglucosamine under alkaline conditions (U.S. Pat. No.5,665,574); methods using N-acetylneuraminic acid aldolase,N-acetylglucosamine 2-epimerase, pyruvic acid and N-acetylglucosamine[Angew. Chem. Int. Ed. Eng., 30, 827 (1991); Carbohydrate Research, 306,575 (1998)]; and methods using N-acetylneuraminic acid synthetase,phosphoenolpyruvic acid and N-acetylmannosamine [Japanese PublishedUnexamined Patent Application No. 4961/98; Glycobiology, 7, 697 (1997)].

The above methods for producing N-acetylneuraminic acid requirecomplicated operations or expensive materials such as pyruvic acid andphosphoenolpyruvic acid, and an economical method for producingN-acetylneuraminic acid has not been established yet.

So far, there has been no report describing or suggesting thatN-acetylneuraminic acid can be produced by utilizing a culture of amicroorganism or a treated matter thereof.

As for the N-acetylneuraminic acid aldolase, those derived from animalsand plants are known, and it is known that microorganisms belonging tothe genus Escherichia have the activity of this enzyme. Also known isthe presence of the gene encoding this enzyme, nanA, in an Escherichiacoli strain [Nucleic Acids Res., 13, 8843 (1985)].

N-Acetylneuraminic acid synthetase is known to be present inmicroorganisms belonging to the genera Escherichia, Neisseria andStreptococcus, etc., and it is known that an Escherichia coli strain hasthe gene encoding this enzyme, neuB [J. Bacteriol., 177, 312 (1995)].

N-Acetylglucosamine 2-epimerase is known to be present in pigs and rats.The properties of the enzyme derived from pig have been investigated[Biochemistry, 17, 3363 (1970)] and the gene encoding the enzyme [J.Biol. Chem., 271, 16294 (1996)] has been obtained. So far, nomicroorganism having the activity of this enzyme is known.

As to the production of pyruvic acid, a process for producing pyruvicacid by using a mutant of Escherichia coli is known [Biosci. Biotech.Biochem., 58, 2164 (1994)].

As to the production of phosphoenolpyruvic acid, a process for producingphosphoenolpyruvic acid by using microorganisms of Saccharomyces, etc.is known (Japanese Published Unexamined Patent Application No.197778/94).

An object of the present invention is to provide a process foreconomically producing N-acetylneuraminic acid without using expensivematerials such as pyruvic acid and phosphoenolpyruvic acid. A furtherobject of the present invention is to provide a process for producingN-acetylneuraminic acid without using expensive N-acetylmannosamine.

SUMMARY OF THE INVENTION

The present inventors have made an intensive investigation to attain theabove objects and have found that N-acetylneuraminic acid can beefficiently produced from inexpensive materials by utilizing amicroorganism which is capable of producing pyruvic acid orphosphoenolpyruvic acid. The present invention has been completed basedon this finding.

The present invention relates to the following (1)-(11).

-   (1) A process for producing N-acetylneuraminic acid which comprises:    -   allowing (i) a culture of a microorganism having        N-acetylneuraminic acid aldolase activity or N-acetylneuraminic        acid synthetase activity, or a treated matter of the        culture, (ii) a culture of a microorganism capable of producing        pyruvic acid or a treated matter of the culture when a        microorganism having N-acetylneuraminic acid aldolase activity        is used in (i) above, or a culture of a microorganism capable of        producing phosphoenolpyruvic acid or a treated matter of the        culture when a microorganism having N-acetylneuraminic acid        synthetase activity is used in (i) above, (iii)        N-acetylmannosamine, and (iv) an energy source which is        necessary for the formation of pyruvic acid or        phosphoenolpyruvic acid to be present in an aqueous medium to        form and accumulate N-acetylneuraminic acid in the aqueous        medium; and    -   recovering N-acetylneuraminic acid from the aqueous medium.-   (2) The process according to the above (1) wherein said    N-acetylmannosamine is produced by allowing a culture of a    microorganism having N-acetylglucosamine 2-epimerase activity or a    treated matter of the culture and N-acetylglucosamine to be present    in an aqueous medium to form and accumulate N-acetylmannosamine in    the aqueous medium.-   (3) The process according to the above (2) wherein said    microorganism having N-acetylglucosamine 2-epimerase activity    carries a recombinant DNA composed of a DNA fragment comprising DNA    encoding N-acetylglucosamine 2-epimerase and a vector.-   (4) The process according to the above (3) wherein said DNA encoding    N-acetylglucosamine 2-epimerase is DNA derived from a microorganism    belonging to the genus Synechocystis.-   (5) The process according to the above (3) or (4) wherein said DNA    encoding N-acetylglucosamine 2-epimerase is selected from the group    consisting of:    -   (a) DNA encoding a protein having the amino acid sequence shown        in SEQ ID NO: 1; and    -   (b) DNA having the nucleotide sequence shown in SEQ ID NO: 2.-   (6) The process according to any of the above (1)-(5) wherein said    microorganism having N-acetylneuraminic acid aldolase activity is a    microorganism belonging to the genus Escherichia or Corynebacterium.-   (7) The process according to any of the above (1)-(6) wherein said    microorganism having N-acetylneuraminic acid synthetase activity is    a microorganism belonging to a genus selected from the group    consisting of Escherichia, Neisseria and Streptococcus.-   (8) The process according to any of the above (1)-(7) wherein said    microorganism capable of producing pyruvic acid is a microorganism    belonging to a genus selected from the group consisting of    Escherichia, Corynebacterium and Saccharomyces.-   (9) The process according to any of the above (1)-(8) wherein said    microorganism capable of producing phosphoenolpyruvic acid is a    microorganism belonging to a genus selected from the group    consisting of Escherichia, Corynebacterium and Saccharomyces.-   (10) The process according to any of the above (6)-(9) wherein said    microorganism belonging to the genus Escherichia is Escherichia    coli.-   (11) The process according to the above (6), (8) or (9) wherein said    microorganism belonging to the genus Corynebacterium is    Corynebacterium ammoniagenes, Corynebacterium glutamicum or    Corynebacterium acetoacidophilum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the steps for constructing plasmid pTA3 expressingN-acetylneuraminic acid aldolase.

FIG. 2 shows the steps for constructing plasmid pYP18 expressingN-acetylneuraminic acid synthetase.

FIG. 3 shows the steps for constructing plasmid PyP16 expressingN-acetylglucosamine 2-epimerase.

EXPLANATION OF SYMBOLS

-   Amp^(r): Ampicillin resistance gene-   P_(L): P_(L) promoter-   cI857: cI857 repressor-   P_(lac): lac promoter-   slrl975: N-Acetylglucosamine 2-epimerase gene-   nanA: N-Acetylneuraminic acid aldolase gene-   neuB: N-Acetylneuraminic acid synthetase gene

DETAILED DESCRIPTION OF THE INVENTION

In the process of the present invention, any microorganism havingN-acetylneuraminic acid aldolase activity can be used. For example,microorganisms belonging to the genus Escherichia or Corynebacterium maybe used.

Examples of the microorganisms belonging to the genus Escherichia arethose of the species Escherichia coli. Examples of the microorganismsbelonging to the genus Corynebacterium are those of the speciesCorynebacterium ammoniagenes, Corynebacterium glutamicum andCorynebacterium acetoacidophilum.

Also useful are transformants with N-acetylneuraminic acid aldolaseactivity enhanced by recombinant DNA techniques. Examples of suchtransformants include microorganisms carrying a recombinant DNAcomprising nanA gene derived from Escherichia coli [Nucleic Acids Res.,13, 8843 (1985)], specifically, Escherichia coli NM522/pTA3.

Any microorganism having N-acetylneuraminic acid synthetase activity canbe used in the process of the present invention. For example,microorganisms belonging to the genus Escherichia, Neisseria orStreptococcus may be used.

Examples of the microorganisms belonging to the genus Escherichia arethose of the species Escherichia coli.

Also useful are transformants with N-acetylneuraminic acid synthetaseactivity enhanced by recombinant DNA techniques. Examples of suchtransformants include microorganisms carrying a recombinant DNAcomprising neuB gene derived from Escherichia coli [J. Bacteriol., 177,312 (1995)], specifically, Escherichia coli NM522/pYP18.

Any microorganism capable of producing pyruvic acid can be used in theprocess of the present invention. Examples of suitable microorganismsare those of the species Escherichia coli, Corynebacterium ammoniagenes,Corynebacterium glutamicum, Corynebacterium acetoacidophilum andSaccharomyces cerevisiae. Also useful are microorganisms with pyruvicacid productivity enhanced by mutagenesis or recombinant DNA techniques.For example, the Escherichia coli mutant described in Biosci. Biotech.Biochem., 58, 2164 (1994) can be used.

Any microorganism capable of producing phosphoenolpyruvic acid can beused in the process of the present invention. Examples of suitablemicroorganisms are those of the species Escherichia coli,Corynebacterium ammoniagenes, Corynebacterium glutamicum,Corynebacterium acetoacidophilum and Saccharomyces cerevisiae. Anexample of the microorganism of the species Saccharomyces cerevisiae isthe strain described in Japanese Published Unexamined Patent ApplicationNo. 197778/94. Also useful are microorganisms with phosphoenolpyruvicacid productivity enhanced by mutagenesis or recombinant DNA techniques.

Any microorganism having N-acetylglucosamine 2-epimerase activity can beused in the process of the present invention. Suitable microorganismsinclude transformants with N-acetylglucosamine 2-epimerase activityenhanced by recombinant DNA techniques. Specific examples of suchtransformants are Escherichia coli carrying the recombinant DNA pEPI1comprising the N-acetylglucosamine 2-epimerase gene derived from pig(FERM BP-4602: U.S. Pat. No. 5,795,767) and Escherichia coli NM522/pYP16carrying a recombinant DNA comprising the N-acetylglucosamine2-epimerase gene derived from a microorganism belonging to the genusSynechocystis.

An example of the N-acetylglucosamine 2-epimerase gene derived from amicroorganism belonging to the genus Synechocystis is the gene encodinga polypeptide having the amino acid sequence shown in SEQ ID NO: 1 whichexists on the chromosome of Synechocystis sp. PCC6803, morespecifically, the gene having the nucleotide sequence shown in SEQ IDNO: 2 (slr1975). The polypeptide having the amino acid sequence shown inSEQ ID NO: 1 and the DNA having the nucleotide sequence shown in SEQ IDNO: 2 have been obtained for the first time by the present inventorsaccording to the procedure described later in an example.

A microorganism having N-acetylneuraminic acid aldolase activity and theability to produce pyruvic acid can be used alone for the production ofN-acetylneuraminic acid from N-acetylmannosamine. In cases where themicroorganism employed is weak or lacking in any of the aboveproperties, it may be used in combination with a microorganism which cancomplement such property for producing N-acetylneuraminic acid.

N-Acetylmannosamine useful in the production of N-acetylneuraminic acidincludes N-acetylmannosamine preparations (e.g., commercial products)and N-acetylmannosamine prepared from N-acetylglucosamine by chemicalreaction under alkaline conditions or by enzymatic conversion usingN-acetylglucosamine 2-epimerase. Also useful are preparations containingN-acetylmannosamine formed and accumulated by allowing a culture of amicroorganism having N-acetylglucosamine 2-epimerase activity or atreated matter of the culture and N-acetylglucosamine to be present inan aqueous medium, and N-acetylmannosamine purified from suchpreparations.

A microorganism having N-acetylneuraminic acid aldolase activity,N-acetylglucosamine 2-epimerase activity and the ability to producepyruvic acid can be used alone for the production of N-acetylneuraminicacid from N-acetylglucosamine. In cases where the microorganism employedis weak or lacking in any of the above properties, it may be used incombination with a microorganism which can complement such property forproducing N-acetylneuraminic acid.

N-Acetylglucosamine useful in the production of N-acetylneuraminic acidincludes N-acetylglucosamine preparations (e.g., commercial products).

A microorganism having N-acetylneuraminic acid synthetase activity andthe ability to produce phosphoenolpyruvic acid can also be used alonefor the production of N-acetylneuraminic acid from N-acetylmannosamine.In cases where the microorganism employed is weak or lacking in any ofthe above properties, it may be used in combination with a microorganismwhich can complement such property for producing N-acetylneuraminicacid.

Further, a microorganism having N-acetylneuraminic acid synthetaseactivity, N-acetylglucosamine 2-epimerase activity and the ability toproduce phosphoenolpyruvic acid can be used alone for the production ofN-acetylneuraminic acid from N-acetylglucosamine. In cases where themicroorganism employed is weak or lacking in any of the aboveproperties, it may be used in combination with a microorganism which cancomplement such property for producing N-acetylneuraminic acid.

The microorganisms employed in the production of N-acetylneuraminic acidor N-acetylmannosamine may be subjected to reaction to form the productduring their growth stage. Alternatively, after the completion ofculturing of a microorganism, the resulting culture or a treated matterof the culture may be subjected to reaction.

As described above, microorganisms prepared by using recombinant DNAtechniques can be used in the production of N-acetylneuraminic acid orN-acetylmannosamine. Gene manipulating operations such as isolation andpurification of plasmid DNA comprising a desired gene from amicroorganism carrying the plasmid, cleavage of plasmid DNA withrestriction enzymes, isolation and purification of cleaved DNAfragments, enzymatic ligation of DNA fragments, and transformation withrecombinant DNA can be carried out according to known methods [e.g.,Molecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989) (hereinafter referred to as MolecularCloning, Second Edition); Current Protocols in Molecular Biology, JohnWiley & Sons (1987-1997) (hereinafter referred to as Current Protocolsin Molecular Biology)]. Polymerase chain reaction (hereinafter referredto as PCR) can be carried out by a known method [PCR Protocols, AcademicPress (1990)].

A gene concerned in the production of N-acetylneuraminic acid orN-acetylmannosamine can be expressed in a host by preparing a DNAfragment of an appropriate length containing the gene from a DNAfragment containing the gene by use of restriction enzymes or PCR,inserting the prepared DNA fragment into an appropriate expressionvector at a site downstream of the promoter, and then introducing theexpression vector comprising the above DNA into a host cell suited forthe expression vector.

As the host cell, any bacterial cells, yeast cells, etc. which arecapable of expressing a desired gene can be used.

The expression vectors that can be employed are those capable ofautonomous replication or integration into chromosome in the above hostcells and comprising a promoter at a position appropriate for thetranscription of a desired DNA.

When a procaryotic cell such as a bacterial cell is used as the hostcell, it is preferred that the expression vector for a gene is arecombinant DNA which is capable of autonomous replication in theprocaryotic cell and which comprises a promoter, a ribosome bindingsequence, the desired DNA, and a transcription termination sequence. Thevector may further comprise a gene regulating the promoter.

Examples of suitable expression vectors are pBTrp2 (Roche Diagnostics),pBTac1 (Roche Diagnostics), pBTac2 (Roche Diagnostics) , pHelix1 (RocheDiagnostics), pKK233-2 (Amersham Pharmacia Biotech), pKK223-3 (AmershamPharmacia Biotech), pGEX-2T (Amersham Pharmacia Biotech), pSE280(Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pQE-30 (QIAGEN), pET-3(Novagen), pKYP10 (Japanese Published Unexamined Patent Application No.110600/83), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric.Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82,4306 (1985)], pBluescript II SK+ (Stratagene), pBluescript IISK-(Stratagene), pTrS30 [prepared from Escherichia coli JM109/pTrS30(FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109/pTrS32(FERM BP-5408)], pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Shuzo Co.,Ltd.), pUC118 (Takara Shuzo Co., Ltd.), pPAC31 (WO 98/12343) and pPA1(Japanese Published Unexamined Patent Application No. 233798/88).

As the promoter, any promoters capable of functioning in host cells suchas Escherichia coli can be used. For example, promoters derived fromEscherichia coli or phage, such as trp promoter (Ptrp), lac promoter(Plac), P_(L) promoter, P_(R) promoter and P_(SE) promoter, SPO1promoter, SPO2 promoter and penP promoter can be used. Artificiallymodified promoters such as a promoter in which two Ptrp are combined intandem, tac promoter, lacT7 promoter and letI promoter, etc. can also beused.

It is preferred to use a plasmid in which the distance between theShine-Dalgarno sequence (ribosome binding sequence) and the initiationcodon is adjusted to an appropriate length (e.g., 6-18 bases).

In the recombinant DNA of the present invention, the transcriptiontermination sequence is not essential for the expression of the desiredDNA, but it is preferred that the transcription termination sequence lieimmediately downstream of the structural gene.

Examples of suitable procaryotes are microorganisms belonging to thegenera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium,Microbacterium and Pseudomonas, specifically, Escherichia coli XL1-Blue,Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coliMC1000, Escherichia coli W1485, Escherichia coli NM522, Escherichia coliJM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coliW3110, Escherichia coli NY49, Serratia ficaria, Serratia fonticola,Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillusamyloliquefaciens, Brevibacterium immariophilum ATCC 14068,Brevibacterium saccharolyticum ATCC 14066, Corynebacterium ammoniagenes,Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC 13869, Corynebacteriumacetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354 andPseudomonas sp. D-0110.

Introduction of the recombinant DNA can be carried out by any of themethods for introducing DNA into the above host cells, for example, themethod using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)],the protoplast method (Japanese Published Unexamined Patent ApplicationNo. 248394/88) and electroporation [Nucleic Acids Research, 16, 6127(1988)].

When a yeast cell is used as the host cell, YEp13 (ATCC 37115), YEp24(ATCC 37051), YCp50 (ATCC 37419), pHS19, pHS15, etc. can be used as theexpression vector.

As the promoter, any promoters capable of functioning in yeast cells canbe used. Suitable promoters include PHO5 promoter, PGK promoter, GAPpromoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shockpolypeptide promoter, MF α 1 promoter, CUP 1 promoter, etc.

Examples of suitable host cells are cells of yeast strains belonging tothe genera Saccharomyces, Schizosaccharomyces, Kluyveromyces,Trichosporon, Schwanniomyces, Pichia and Candida, specifically,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastorisand Candida utilis.

Introduction of the recombinant DNA can be carried out by any of themethods for introducing DNA into yeast cells, for example,electroporation [Methods in Enzymol., 194, 182 (1990)], the spheroplastmethod [Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)] and the lithiumacetate method [J. Bacteriol., 153, 163 (1983)].

Culturing of the transformant of the present invention can be carriedout by conventional methods for culturing the host cell of thetransformant.

For the culturing of the transformant prepared by using a procaryoticcell such as Escherichia coli cell or a eucaryotic cell such as a yeastcell as the host cell, any of natural media and synthetic media can beused insofar as it is a medium suitable for efficient culturing of thetransformant which contains carbon sources, nitrogen sources, inorganicsubstances, etc. which can be assimilated by the host used.

As the carbon sources, any carbon sources which can be assimilated bythe host can be used. Examples of suitable carbon sources includecarbohydrates such as glucose, fructose, sucrose, molasses containingthem, starch and starch hydrolyzate; organic acids such as acetic acidand propionic acid; and alcohols such as ethanol and propanol.

As the nitrogen sources, ammonia, ammonium salts of inorganic or organicacids such as ammonium chloride, ammonium sulfate, ammonium acetate andammonium phosphate, and other nitrogen-containing compounds can be usedas well as peptone, meat extract, yeast extract, corn steep liquor,casein hydrolyzate, soybean cake, soybean cake hydrolyzate, and variousfermented cells and digested products thereof.

Examples of the inorganic substances include potassiumdihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate and calcium carbonate.

Culturing is usually carried out under aerobic conditions, for example,by shaking culture or submerged spinner culture under aeration, at15-40° C. for 5 hours to 7 days. The pH is maintained at 3.0-9.0 duringthe culturing. The pH adjustment is carried out by using an inorganic ororganic acid, an alkali solution, urea, calcium carbonate, ammonia, etc.

If necessary, antibiotics such as ampicillin and tetracycline may beadded to the medium during the culturing.

When a microorganism transformed with an expression vector comprising aninducible promoter is cultured, an inducer may be added to the medium,if necessary. For example, in the case of a microorganism transformedwith an expression vector comprising lac promoter,isopropyl-β-D-thiogalactopyranoside or the like may be added to themedium; and in the case of a microorganism transformed with anexpression vector comprising trp promoter, indoleacrylic acid or thelike may be added.

The treated matters of a culture include concentrated culture, driedculture, cells obtained by centrifuging the culture, products obtainedby treating the cells by various means such as drying, freeze-drying,treatment with a surfactant, ultrasonication, mechanical friction,treatment with a solvent, enzymatic treatment, protein fractionation andimmobilization, an enzyme preparation obtained by extracting the cells,etc.

The amount of each microorganism used in the production ofN-acetylneuraminic acid or N-acetylmannosamine is 1-500 g/l, preferably1-300 g/l, as wet cells.

As the energy source which is necessary for the formation of pyruvicacid or phosphoenolpyruvic acid, any substance can be used that promotesthe formation of pyruvic acid or phosphoenolpyruvic acid. Examples ofpreferred substances are glucose and fructose. The substances may beused usually at a concentration of 10-300 g/l.

Aqueous media useful in the production of N-acetylneuraminic acid orN-acetylmannosamine include water, buffers such as phosphate buffer,carbonate buffer, acetate buffer, borate buffer, citrate buffer and Trisbuffer, alcohols such as methanol and ethanol, esters such as ethylacetate, ketones such as acetone, amides such as acetamide, etc. Alsouseful is a medium or culture broth of a microorganism used in theprocess for producing N-acetylneuraminic acid or N-acetylmannosamine.

If necessary, a chelating agent (e.g., phytic acid), a surfactant or anorganic solvent may be added in the process for producingN-acetylneuraminic acid or N-acetylmannosamine.

Any surfactant that promotes the formation of N-acetylneuraminic acid orN-acetylmannosamine can be used. Suitable surfactants include nonionicsurfactants such as polyoxyethylene octadecylamine (e.g., Nymeen S-215,NOF Corporation), cationic surfactants such as cetyltrimethylammoniumbromide and alkyldimethyl benzylammonlum chloride (e.g., Cation F2-40E,NOF Corporation), anionic surfactants such as lauroyl sarcosinate, andtertiary amines such as alkyldimethylamine (e.g., Tertiary Amine FB, NOFCorporation), which may be used alone or in combination. The surfactantis usually used at a concentration of 0.1-50 g/l.

As the organic solvent, xylene, toluene, aliphatic alcohols, acetone,ethyl acetate, etc. may be used usually at a concentration of 0.1-50ml/l.

The reaction for forming N-acetylneuraminic acid or N-acetylmannosamineis carried out in an aqueous medium at pH 5-10, preferably pH 6-8, at20-50° C. for 1-96 hours.

Adenine, adenosine 5′-monotriphosphate (AMP), adenosine 5′-triphosphate(ATP), magnesium sulfate, magnesium chloride, etc. may be added forpromoting the reaction. Adenine, AMP and ATP are usually used at aconcentration of 0.01-100 mmol/l.

N-Acetylneuraminic acid or N-acetylmannosamine formed in the aqueousmedium can be determined by using a carbohydrate analysis system(Dionex) or the like [Anal. Biochem., 189, 151 (1990)].

N-Acetylneuraminic acid or N-acetylmannosamine can be recovered from thereaction mixture by ordinary methods using active carbon, ion-exchangeresins, etc.

Certain embodiments of the present invention are illustrated in thefollowing examples. These examples are not to be construed as limitingthe scope of the invention.

EXAMPLE 1

Construction of a Strain Expressing N-Acetylneuraminic Acid AldolaseGene Derived from Escherichia coli

Escherichia coli W3110 (ATCC 27325) was cultured by the method describedin Current Protocols in Molecular Biology and the chromosomal DNA of themicroorganism was isolated and purified.

The DNA primer shown in SEQ ID NO: 3 and the DNA primer shown in SEQ IDNO: 4 were synthesized by using a DNA synthesizer (Model 8905,PerSeptive Biosystems).

PCR was carried out using the above synthetic DNAs as primers and thechromosomal DNA of Escherichia coli W3110 (ATCC 27325) as a template.That is, PCR was carried out by 30 cycles, one cycle consisting ofreaction at 94° C. for one minute, reaction at 42° C. for 2 minutes andreaction at 72° C. for 3 minutes, using 40 μl of a reaction mixturecomprising 0.1 μg of the chromosomal DNA, 0.5 μmol/l each of theprimers, 2.5 units of Pfu DNA polymerase (Stratagene), 4 μl of bufferfor Pfu DNA polymerase (10×) (Stratagene) and 200 μmol/l each ofdeoxyNTPs.

One-tenth of the resulting reaction mixture was subjected to agarose gelelectrophoresis to confirm that the desired fragment was amplified.Then, the remaining reaction mixture was mixed with an equal amount ofphenol/chloroform (1 vol/1 vol) saturated with TE [10 mmol/l Tris-HCl(pH 8.0), 1 mmol/l EDTA], followed by centrifugation. The obtained upperlayer was mixed with a two-fold volume of cold ethanol and allowed tostand at −80° C. for 30 minutes. The resulting mixture was centrifugedto obtain a DNA precipitate.

The DNA precipitate was dissolved in 20 μl of TE and 5 μl of thesolution was subjected to reaction to cleave the DNA with restrictionenzymes HindIII and BamHI. DNA fragments were separated by agarose gelelectrophoresis and a 1.2 kb fragment was recovered using Gene Clean IIKit (Bio 101). pUC19 DNA [Gene, 33, 103 (1985)] (0.2 μg) was cleavedwith restriction enzymes HindIII and BamHI. DNA fragments were separatedby agarose gel electrophoresis and a 2.7 kb fragment was recovered inthe same manner.

The 1.2 kb fragment and 2.7 kb fragment obtained above were subjected toligation reaction using a ligation kit at 16° C. for 16 hours.Escherichia coli NM522 capable of producing pyruvic acid was transformedusing the ligation mixture according to the known method describedabove, spread on LB agar medium containing 50 μg/ml ampicillin, andcultured overnight at 30° C.

Escherichia coli NM522/pTA3, which is a transformant carrying theN-acetylneuraminic acid aldolase gene, nanA, was obtained from a colonyof the transformant that grew on the above medium. A plasmid wasextracted from this transformant by a known method to obtain pTA3, whichis a plasmid for expression of N-acetylneuraminic acid aldolase gene.The structure of this plasmid was confirmed by digestion withrestriction enzymes (FIG. 1).

EXAMPLE 2

Production of N-Acetylneuraminic Acid

Escherichia coli NM522/pTA3 obtained in Example 1 was inoculated into125 ml of LB medium containing 50 μg/ml ampicillin in a 1-l Erlenmeyerflask with baffles, followed by culturing at 28° C. with stirring (220r.p.m.) for 17 hours. The resulting culture (125 ml) was inoculated into2.5 l of a liquid medium (pH unadjusted) comprising 10 g/l glucose, 12g/l Bacto-tryptone (Difco Laboratories Inc.), 24 g/l yeast extract(Difco Laboratories Inc.), 2.3 g/l KH₂PO₄, 12.5 g/l K₂HPO₄ and 50 μg/mlampicillin in a 5-l jar fermentor. Culturing was carried out at 37° C.for 6 hours under the conditions of stirring at 600 r.p.m. and aerationat 2.5 l/min. During the culturing, the pH of culture was maintained at7.0 with 28% aqueous ammonia. Glucose was added, according to need, inthe course of culturing. The resulting culture was centrifuged to obtainwet cells. The wet cells could be stored at −20° C. and could be usedafter thawing, according to need.

A reaction mixture (30 ml) comprising 50 g/l Escherichia coli NM522/pTA3wet cells, 65 g/l fructose, 40 g/l N-acetylmannosamine, 25 g/l KH₂PO₄, 5g/l MgSO₄.7H₂O, 5 g/l phytic acid, 4 g/l Nymeen S-215 and 10 ml/l xylenewas put into a 200-ml beaker and subjected to reaction at 32° C. for 25hours with stirring (900 r.p.m.) using a magnetic stirrer. During thereaction, the pH of reaction mixture was maintained at 7.2 with 4 NNaOH, and according to need, fructose and KH₂PO₄ were added to thereaction mixture.

After the completion of reaction, the reaction product was analyzed byusing a carbohydrate analysis system (DX-500, Dionex) and it was foundthat 0.34 g/l N-acetylneuraminic acid was formed and accumulated in thereaction mixture.

EXAMPLE 3

Construction of a Strain Expressing N-Acetylneuraminic Acid SynthetaseGene Derived from Escherichia coli

Escherichia coli K235 (ATCC 13027) was cultured by the method describedin Current Protocols in Molecular Biology and the chromosomal DNA of themicroorganism was isolated and purified.

The DNA primer shown in SEQ ID NO: 5 and the DNA primer shown in SEQ IDNO: 6 were synthesized by using a DNA synthesizer (Model 8905,PerSeptive Biosystems).

PCR was carried out using the above synthetic DNAs as primers and thechromosomal DNA of Escherichia coli K235 (ATCC 13027) as a template.That is, PCR was carried out by 30 cycles, one cycle consisting ofreaction at 94° C. for one minute, reaction at 42° C. for 2 minutes andreaction at 72° C. for 3 minutes, using 40 μl of a reaction mixturecomprising 0.1 μg of the chromosomal DNA, 0.5 μmol/l each of theprimers, 2.5 units of Pfu DNA polymerase (Stratagene), 4 μl of bufferfor Pfu DNA polymerase (10×) (Stratagene) and 200 μmol/l each ofdeoxyNTPs.

One-tenth of the resulting reaction mixture was subjected to agarose gelelectrophoresis to confirm that the desired fragment was amplified.Then, the remaining reaction mixture was mixed with an equal amount ofphenol/chloroform (1 vol/1 vol) saturated with TE, followed bycentrifugation. The obtained upper layer was mixed with a two-foldvolume of cold ethanol and allowed to stand at −80° C. for 30 minutes.The resulting mixture was centrifuged to obtain a DNA precipitate.

The DNA precipitate was dissolved in 20 μl of TE and 5 μl of thesolution was subjected to reaction to cleave the DNA with restrictionenzymes ClaI and BamHI. DNA fragments were separated by agarose gelelectrophoresis and a 1.1 kb fragment was recovered using Gene Clean IIKit (Bio 101). pPAC31 DNA (WO98/12343) (0.2 μg) was cleaved withrestriction enzymes ClaI and BamHI. DNA fragments were separated byagarose gel electrophoresis and a 5.5 kb fragment was recovered in thesame manner.

The 1.1 kb fragment and 5.5 kb fragment obtained above were subjected toligation reaction using a ligation kit at 16° C. for 16 hours.Escherichia coli NM522 capable of producing phosphoenolpyruvic acid wastransformed using the ligation mixture according to the known methoddescribed above, spread on LB agar medium containing 50 μg/mlampicillin, and cultured overnight at 30° C.

Escherichia coli NM522/pYP18, which is a transformant carrying theN-acetylneuraminic acid synthetase gene, neuB, was obtained from acolony of the transformant that grew on the above medium. A plasmid wasextracted from this transformant by a known method to obtain pYP18,which is a plasmid for expression of N-acetylneuraminic acid synthetasegene. The structure of this plasmid was confirmed by digestion withrestriction enzymes (FIG. 2).

EXAMPLE 4

Production of N-Acetylneuraminic Acid

Escherichia coli NM522/pYP18 obtained in Example 3 was inoculated into125 ml of LB medium containing 50 μg/ml ampicillin in a 1-l Erlenmeyerflask with baffles, followed by culturing at 28° C. with stirring (220r.p.m.) for 17 hours. The resulting culture (125 ml) was inoculated into2.5 l of a liquid medium (pH unadjusted) comprising 10 g/l glucose, 12g/l Bacto-tryptone (Difco Laboratories Inc.), 24 g/l yeast extract(Difco Laboratories Inc.), 2.3 g/l KH₂PO₄, 12.5 g/l K₂HPO₄ and 50 μg/mlampicillin in a 5-l jar fermentor. Culturing was carried out at 37° C.for 4 hours and then at 40° C. for 3 hours, under the conditions ofstirring at 600 r.p.m. and aeration at 2.5 l/min. During the culturing,the pH of culture was maintained at 7.0 with 28% aqueous ammonia.Glucose was added, according to need, in the course of culturing.

The resulting culture was centrifuged to obtain wet cells. The wet cellscould be stored at −20° C. and could be used after thawing, according toneed.

A reaction mixture (30 ml) comprising 50 g/l Escherichia coliNM522/pYP18 wet cells, 65 g/l fructose, 40 g/l N-acetylmannosamine, 25g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phytic acid, 4 g/l Nymeen S-215 and10 ml/l xylene was put into a 200-ml beaker and subjected to reaction at32° C. for 19 hours with stirring (900 r.p.m.) using a magnetic stirrer.During the reaction, the pH of reaction mixture was maintained at 7.2with 4 N NaOH, and according to need, fructose and KH₂PO₄ were added tothe reaction mixture.

After the completion of reaction, the reaction product was analyzed byusing a carbohydrate analysis system (DX-500, Dionex) and it was foundthat 1.4 g/l N-acetylneuraminic acid was formed and accumulated in thereaction mixture.

EXAMPLE 5

Production of N-Acetylneuraminic Acid

Escherichia coli NM522/pYP18 obtained in Example 3 was culturedaccording to the method described in Example 2 and the resulting culturewas centrifuged to obtain wet cells. The wet cells could be stored at−20° C. and could be used after thawing, according to need.

Corynebacterium ammoniagenes ATCC 21170 was inoculated into 25 ml of aliquid medium comprising 50 g/l glucose, 10 g/l polypeptone (NihonPharmaceutical Industrial Co., Ltd.), 10 g/l yeast extract (OrientalYeast Co., Ltd.), 5 g/l urea, 5 g/l (NH₄)₂SO₄, 1 g/l KH₂PO₄, 3 g/lK₂HPO₄, 1 g/l MgSO₄.7H₂O, 0.1 g/l CaCl₂.2H₂O, 10 mg/l FeSO₄.7H₂O, 10mg/l ZnSO₄.7H₂O, 20 mg/l MnSO₄.4-6H₂O, 20 mg/l L-cysteine, 10 mg/lcalcium D-pantothenate, 5 mg/l vitamin B₁, 5 mg/l nicotinic acid and 30μg/l biotin (adjusted to pH 7.2 with 10 N NaOH) in a 300-ml Erlenmeyerflask with baffles, followed by culturing at 28° C. with stirring (220r.p.m.) for 24 hours.

The resulting culture (20 ml) was inoculated into 250 ml of a liquidmedium having the same composition as above in a 2-l Erlenmeyer flaskwith baffles, followed by culturing at 28° C. with stirring (220 r.p.m.)for 24 hours. The obtained culture was used as a seed culture.

The seed culture (250 ml) was inoculated into 2.25 l of a liquid mediumcomprising 150 g/l glucose, 5 g/l meat extract (Kyokuto PharmaceuticalInd. Co., Ltd.), 10 g/l KH₂PO₄, 10 g/l K₂HPO₄, 10 g/l MgSO₄.7H₂O, 0.1g/l CaCl₂.2H₂O, 20 mg/l FeSO₄.7H₂O, 10 mg/l ZnSO₄.7H₂O, 20 mg/lMnSO₄.4-6H₂O (separately sterilized), 15 mg/l β-alanine (separatelysterilized), 20 mg/l L-cysteine, 100 μg/l biotin, 2 g/l urea and 5 mg/lvitamin B₁ (separately sterilized) (adjusted to pH 7.2 with 10 N NaOH)in a 5-l jar fermentor. Culturing was carried out at 32° C. for 24 hoursunder the conditions of stirring at 600 r.p.m. and aeration at 2.5l/min. During the culturing, the pH of culture was maintained at 6.8with 28% aqueous ammonia.

The resulting culture was centrifuged to obtain wet cells. The wet cellscould be stored at −20° C. and could be used after thawing, according toneed.

A reaction mixture (30 ml) comprising 50 g/l Escherichia coliNM522/pYP18 wet cells, 150 g/l Corynebacterium ammoniagenes ATCC 21170wet cells, 65 g/l fructose, 40 g/l N-acetylmannosamine, 25 g/l KH₂PO₄, 5g/l MgSO₄.7H₂O, 5 g/l phytic acid, 4 g/l Nymeen S-215 and 10 ml/l xylenewas put into a 200-ml beaker and subjected to reaction at 32° C. for 6hours with stirring (900 r.p.m.) using a magnetic stirrer. During thereaction, the pH of reaction mixture was maintained at 7.2 with 4 NNaOH, and according to need, fructose and KH₂PO₄ were added to thereaction mixture.

After the completion of reaction, the reaction product was analyzed byusing a carbohydrate analysis system (DX-500, Dionex) and it was foundthat 3.1 g/l N-acetylneuraminic acid was formed and accumulated in thereaction mixture.

EXAMPLE 6

Construction of a Strain Expressing N-Acetylglucosamine 2-Epimerase GeneDerived from Synechocystis

Blast Search of Genbank and a similarity search on CyanoBase(http://www.kazusa.or.jp/cyano/), which is a database of the genomicsequence of Synechocystis sp. (PCC6803), were conducted with the aminoacid sequence of N-acetylglucosamine 2-epimerase derived from pig [J.Biol. Chem., 271, 16294 (1996)] as a query. As a result, the above aminoacid sequence showed a high homology to the sequence derived fromSynechocystis sp. (PCC6803) described as a renin-binding protein(slr1975).

Synechocystis sp. (PCC6803) was cultured by the method described in J.Gen. Microbiol., 111, 1 (1979), and the chromosomal DNA of themicroorganism was isolated and purified by the method described inCurrent Protocols in Molecular Biology.

PCR was carried out according to the method described in Example 1 usingthe DNAs shown in SEQ ID NOS: 7 and 8 which had been synthesized byusing a DNA synthesizer (Model 8905, PerSeptive Biosystems) as primersand the chromosomal DNA of Synechocystis sp. (PCC6803) as a template.

One-tenth of the resulting reaction mixture was subjected to agarose gelelectrophoresis to confirm that the desired fragment was amplified.Then, the remaining reaction mixture was mixed with an equal amount ofphenol/chloroform (1 vol/1 vol) saturated with TE.

The resulting mixture was centrifuged and the obtained upper layer wasmixed with a two-fold volume of cold ethanol and allowed to stand at−80° C. for 30 minutes. The resulting mixture was centrifuged to obtaina DNA precipitate.

The DNA precipitate was dissolved in 20 μl of TE and 5 μl of thesolution was subjected to reaction to cleave the DNA with restrictionenzymes ClaI and BamHI. DNA fragments were separated by agarose gelelectrophoresis and a 1.2 kb fragment was recovered using Gene Clean IIKit (Bio 101).

pPAC31 DNA (0.2 μg) was cleaved with restriction enzymes ClaI and BamHI.DNA fragments were separated by agarose gel electrophoresis and a 5.5 kbfragment was recovered in the same manner.

The 1.2 kb fragment and 5.5 kb fragment obtained above were subjected toligation reaction using a ligation kit at 16° C. for 16 hours.

Escherichia coli NM522 was transformed using the ligation mixtureaccording to the known method described above, spread on LB agar mediumcontaining 50 μg/ml ampicillin, and cultured overnight at 30° C.

Escherichia coli NM522/pYP16, which is a transformant carrying the DNAencoding N-acetylglucosamine 2-epimerase derived from Synechocystis sp.,was obtained from a colony of the transformant that grew on the abovemedium. A plasmid was extracted from this transformant by a known methodto obtain expression plasmid pYP16. The structure of this plasmid wasconfirmed by digestion with restriction enzymes (FIG. 3).

EXAMPLE 7

Production of N-Acetylneuraminic Acid

Escherichia coli NM522/pTA3 obtained in Example 1 was cultured accordingto the method described in Example 2 and the resulting culture wascentrifuged to obtain wet cells. The wet cells could be stored at −20°C. and could be used after thawing, according to need.

Escherichia coli NM522/pYP16 obtained in Example 6 was inoculated into125 ml of LB medium containing 50 μg/ml ampicillin in a 1-l Erlenmeyerflask with baffles, followed by culturing at 28° C. with stirring (220r.p.m.) for 17 hours. The resulting culture (125 ml) was inoculated into2.5 l of a liquid medium (pH unadjusted) comprising 10 g/l glucose, 12g/l Bacto-tryptone (Difco Laboratories Inc.), 24 g/l yeast extract(Difco Laboratories Inc.), 2.3 g/l KH₂PO₄, 12.5 g/l K₂HPO₄ and 50 μg/mlampicillin in a 5-l jar fermentor. Culturing was carried out at 30° C.for 4 hours and then at 40° C. for 3 hours, under the conditions ofstirring at 600 r.p.m. and aeration at 2.5 l/min. During the culturing,the pH of culture was maintained at 7.0 with 28% aqueous ammonia.Glucose was added, according to need, in the course of culturing.

The resulting culture was centrifuged to obtain wet cells. The wet cellscould be stored at −20° C. and could be used after thawing, according toneed.

Corynebacterium ammoniagenes ATCC 21170 was cultured according to themethod described in Example 5 and the resulting culture was centrifugedto obtain wet cells. The wet cells could be stored at −20° C. and couldbe used after thawing, according to need.

A reaction mixture (30 ml) comprising 50 g/l Escherichia coli NM522/pTA3wet cells, 50 g/l Escherichia coli NM522/pYP16 wet cells, 150 g/lCorynebacterium ammoniagenes ATCC 21170 wet cells, 65 g/l fructose, 180g/l N-acetylglucosamine, 25 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O, 5 g/l phyticacid, 4 g/l Nymeen S-215 and 10 ml/l xylene was put into a 200-ml beakerand subjected to reaction at 32° C. for 24 hours with stirring (900r.p.m.) using a magnetic stirrer. During the reaction, the pH ofreaction mixture was maintained at 7.2 with 4 N NaOH, and according toneed, fructose and KH₂PO₄ were added to the reaction mixture.

After the completion of reaction, the reaction product was analyzed byusing a carbohydrate analysis system (DX-500, Dionex) and it was foundthat 1.0 g/l N-acetylneuraminic acid was formed and accumulated in thereaction mixture.

EXAMPLE 8

Production of N-Acetylneuraminic Acid

Escherichia coli NM522/pYP18 obtained in Example 3 and Escherichia coliNM522/pYP16 obtained in Example 6 were cultured according to the methodsdescribed in Examples 4 and 7, respectively, and the resulting cultureswere centrifuged to obtain wet cells. The wet cells could be stored at−20° C. and could be used after thawing, according to need.

A reaction mixture (30 ml) comprising 50 g/l Escherichia coliNM522/pYP16 wet cells, 50 g/l Escherichia coli NM522/pYP18 wet cells, 65g/l fructose, 180 g/l N-acetylglucosamine, 25 g/l KH₂PO₄, 5 g/lMgSO₄.7H₂O, 5 g/l phytic acid, 4 g/l Nymeen S-215 and 10 ml/l xylene wasput into a 200-ml beaker and subjected to reaction at 32° C. for 11hours with stirring (900 r.p.m.) using a magnetic stirrer. During thereaction, the pH of reaction mixture was maintained at 7.2 with 4 NNaOH, and according to need, fructose and KH₂PO₄ were added to thereaction mixture.

After the completion of reaction, the reaction product was analyzed byusing a carbohydrate analysis system (DX-500, Dionex) and it was foundthat 1.3 g/l N-acetylneuraminic acid was formed and accumulated in thereaction mixture.

EXAMPLE 9

Production of N-Acetylneuraminic Acid

Escherichia coli NM522/pYP18 obtained in Example 3 and Escherichia coliNM522/pYP16 obtained in Example 6 were cultured according to the methodsdescribed in Examples 4 and 7, respectively, and the resulting cultureswere centrifuged to obtain wet cells. The wet cells could be stored at−20° C. and could be used after thawing, according to need.

Corynebacterium ammoniagenes ATCC 21170 was cultured according to themethod described in Example 5 and the resulting culture was centrifugedto obtain wet cells. The wet cells could be stored at −20° C. and couldbe used after thawing, according to need.

A reaction mixture (30 ml) comprising 50 g/l Escherichia coliNM522/pYP16 wet cells, 50 g/l Escherichia coli NM522/pYP18 wet cells,150 g/l Corynebacterium ammoniagenes ATCC 21170 wet cells, 65 g/lfructose, 180 g/l N-acetylglucosamine, 25 g/l KH₂PO₄, 5 g/l MgSO₄.7H₂O,5 g/l phytic acid, 4 g/l Nymeen S-215 and 10 ml/l xylene was put into a200-ml beaker and subjected to reaction at 32° C. for 24 hours withstirring (900 r.p.m.) using a magnetic stirrer. During the reaction, thepH of reaction mixture was maintained at 7.2 with 4 N NaOH, andaccording to need, fructose and KH₂PO₄ were added to the reactionmixture.

After the completion of reaction, the reaction product was analyzed byusing a carbohydrate analysis system (DX-500, Dionex) and it was foundthat 4.3 g/l N-acetylneuraminic acid was formed and accumulated in thereaction mixture.

EXAMPLE 10

Production of N-Acetylneuraminic Acid

Escherichia coli NM522/pYP18 obtained in Example 3 and Escherichia coliNM522/pYP16 obtained in Example 6 were cultured according to the methodsdescribed in Examples 4 and 7, respectively, and the resulting cultureswere centrifuged to obtain wet cells. The wet cells could be stored at−20° C. and could be used after thawing, according to need.

Corynebacterium ammoniagenes ATCC 21170 was cultured according to themethod described in Example 5 and the resulting culture was centrifugedto obtain wet cells. The wet cells could be stored at −20° C. and couldbe used after thawing, according to need.

A reaction mixture (30 ml) comprising 50 g/l Escherichia coliNM522/pYP16 wet cells, 50 g/l Escherichia coli NM522/pYP18 wet cells,150 g/l Corynebacterium ammoniagenes ATCC 21170 wet cells, 100 g/lglucose, 180 g/l N-acetylglucosamine, 5 g/l adenine, 15 g/l KH₂PO₄, 5g/l MgSO₄.7H₂O, 5 g/l phytic acid, 4 g/l Nymeen S-215 and 10 ml/l xylenewas put into a 200-ml beaker and subjected to reaction at 32° C. for 22hours with stirring (900 r.p.m.) using a magnetic stirrer. During thereaction, the pH of reaction mixture was maintained at 7.2 with 4 NNaOH, and according to need, glucose and KH₂PO₄ were added to thereaction mixture.

After the completion of reaction, the reaction product was analyzed byusing a carbohydrate analysis system (DX-500, Dionex) and it was foundthat 12.3 g/l N-acetylneuraminic acid was formed and accumulated in thereaction mixture.

1. A process for producing N-acetylneuraminic acid which comprises:providing, in an aqueous medium (i) a culture of a microorganism havingN-acetylneuraminic acid aldolase activity or N-acetylneuraminic acidsynthetase activity; (ii) a culture of a microorganism capable ofproducing pyruvic acid when a culture of a microorganism havingN-acetylneuraminic acid aldolase activity is used in (i), or a cultureof a microorganism capable of producing phosphoenolpyruvic acid when aculture of a microorganism having N-acetylneuraminic acid synthetaseactivity is used in (i); (iii) N-acetylmannosamine which is produced byallowing a culture of a microorganism harboring DNA encodingN-acetylglucosamine 2-epimerase isolated from a microorganism belongingto the genus Synechocystis, and N-acetylglucosamine to be present in theaqueous medium to form and accumulate N-acetylmannosamine in the culturemedium, and (iv) an energy source which is necessary for the formationof a pyruvic acid or phosphoenolpyruvic acid; allowingN-acetylneuraminic acid to form and accumulate in the aqueous medium;and recovering N-acetylneuraminic acid from the aqueous medium whereinsaid cultures are independently provided as cultures per se or treatedmatters thereof, wherein said treated matters are selected from thegroup consisting of concentrated culture, dried culture, cells obtainedby centrifuging the culture, products obtained by treating the cells bydrying, freeze-drying, ultrasonication, mechanical friction, proteinfractionation and cell-immobilization, and an enzyme preparationobtained by extracting the cells, or a preparation obtained by treatmentwith enzymes, surfactant or solvent, and wherein said treated mattercontinues to have said enzyme activity.
 2. A process for producingN-acetylneuraminic acid which comprises: providing, in aqueous medium(i) a culture of a microorganism having N-acetylneuraminic acid aldolaseactivity or N-acetylneuraminic acid synthetase activity; (ii) a cultureof a microorganism capable of producing pyruvic acid when a culture of amicroorganism having N-acetylneuraminic acid aldolase activity is usedin (i), or a culture of a microorganism capable or producingphosphoenolpyruvic acid when a culture of a microorganism havingN-acetylneuraminic acid synthetase activity is used in (i); (iii)N-acetylmannosamine which is produced by allowing a culture of amicroorganism harboring DNA comprising SEQ ID NO:2 encodingN-acetylglucosamine 2-epimerase comprising SEQ ID NO:1 and being capableof forming N-acetylmannosamine from N-acetylglucosamine so as to formand accumulate N-acetylmannosamine in the aqueous medium; and (iv) anenergy source which is necessary for the formation of pyruvic acid orphosphoenolpyruvic acid; allowing N-acetylneuraminic acid to form andaccumulate in the aqueous medium; and recovering N-acetylneuraminic acidfrom the aqueous medium wherein said cultures are independently providedas cultures per se or treated matters thereof, wherein said treatedmatters are selected from the group consisting of concentrated culture,dried culture, cells obtained by centrifuging the culture, productsobtained by treating the cells by drying, freeze-drying,ultrasonication, mechanical friction, protein fractionation andcell-immobilization, and an enzyme preparation obtained by extractingthe cells, or a preparation obtained by treatment with enzymes,surfactant or solvent, and wherein said treated matter continues to havesaid enzyme activity.
 3. A process for producing N-acetylneuraminic acidwhich comprises: providing, in an aqueous medium (i) a culture of amicroorganism having N-acetylneuraminic acid aldolase activity; (ii) aculture of a microorganism capable of producing pyruvic acid; (iii)N-acetylmannosamine which is produced by allowing a culture of amicroorganism harboring DNA selected from the group consisting of (a)DNA encoding a protein comprising the amino acid sequence shown in SEQID NO: 1 and being capable of forming N-acetylmannosamine fromN-acetylglucosamine; and (b) DNA comprising the nucleotide sequenceshown in SEQ ID NO: 2 encoding N-acetylglucosamine 2-epimerasecomprising SEQ ID NO:1 and being capable of forming N-acetylmannosaminefrom N-acetylglucosamine so as to form and accumulateN-acetylmannosamine in the aqueous medium; and (iv) an energy sourcewhich is necessary for the formation of pyruvic acid; allowingN-acetylneuraminic acid to form and accumulate in the aqueous medium;and recovering N-acetylneuraminic acid from the aqueous medium whereinsaid cultures are independently provided as cultures per se or treatedmatters thereof, wherein said treated matters are selected from thegroup consisting of concentrated culture, dried culture, cells obtainedby centrifuging the culture, products obtained by treating the cells bydrying, freeze-drying, ultrasonication, mechanical friction, proteinfractionation and cell-immobilization, and an enzyme preparationobtained by extracting the cells, or a preparation obtained by treatmentwith enzymes, surfactant or solvent, and wherein said treated mattercontinues to have said enzyme activity.
 4. A process for producingN-acetylneuraminic acid which comprises: providing, in an aqueous medium(i) a culture of a microorganism having N-acetylneuraminic acidsynthetase activity; (ii) a culture of a microorganism capable ofproducing phosphoenolpyruvic acid; (iii) N-acetylmannosamine which isproduced by allowing a culture of a microorganism harboring DNA selectedfrom the group consisting of (a) DNA encoding a protein comprising theamino acid sequence shown in SEQ ID NO: 1 and being capable of formingN-acetylmannosamine from N-acetylglucosamine; and (b) DNA comprising thenucleotide sequence shown in SEQ ID NO: 2 encoding N-acetylglucosamine2-epimerase comprising SEQ ID NO:1 and being capable of formingN-acetylmannosamine from N-acetylglucosamine so as to form andaccumulate N-acetylmannosamine in the aqueous medium; and (iv) an energysource which is necessary for the formation of phosphoenolpyruvic acid;allowing N-acetylneuraminic acid to form and accumulate in the aqueousmedium; and recovering N-acetylneuraminic acid from the aqueous mediumwherein said cultures are independently provided as cultures per se ortreated matters thereof, wherein said treated matters are selected fromthe group consisting of concentrated culture, dried culture, cellsobtained by centrifuging the culture, products obtained by treating thecells by drying, freeze-drying, ultrasonication, mechanical friction,protein fractionation and cell-immobilization, and an enzyme preparationobtained by extracting the cells, or a preparation obtained by treatmentwith enzymes, surfactant or solvent, and wherein said treated mattercontinues to have said enzyme activity.
 5. A process for producingN-acetylneuraminic acid which comprises: providing, in an aqueous medium(i) a culture of a microorganism having N-acetylneuraminic acid aldolaseactivity; (ii) a culture of a microorganism capable of producing pyruvicacid; (iii) a culture of a microorganism harboring DNA encodingN-acetylglucosamine 2-epimerase isolated from a microorganism belongingto the genus Synechocystis; (iv) an energy source which is necessary forthe formation of pyruvic acid; and (v) N-acetylglucosamine; allowingN-acetylneuraminic acid to form and accumulate in the aqueous medium;and recovering N-acetylneuraminic acid from the aqueous medium whereinsaid cultures are independently provided as cultures per se or treatedmatters thereof, wherein said treated matters are selected from thegroup consisting of concentrated culture, dried culture, cells obtainedby centrifuging the culture, products obtained by treating the cells bydrying, freeze-drying, ultrasonication, mechanical friction, proteinfractionation and cell-immobilization, and an enzyme preparationobtained by extracting the cells, or a preparation obtained by treatmentwith enzymes, surfactant or solvent, and wherein said treated mattercontinues to have said enzyme activity.
 6. A process for producingN-acetylneuraminic acid which comprises: providing, in an aqueous medium(i) a culture of a microorganism having N-acetylneuraminic acidsynthetase activity; (ii) a culture of a microorganism capable ofproducing phosphoenolpyruvic acid; (iii) a culture of a microorganismharboring DNA encoding N-acetylglucosamine 2-epimerase isolated from amicroorganism belonging to the genus Synechocystis; (iv) an energysource which is necessary for the formation of phosphoenolpyruvic acid;and (v) N-acetylglucosamine; allowing N-acetylneuraminic acid to formand accumulate in the aqueous medium; and recovering N-acetylneuraminicacid from the aqueous medium wherein said cultures are independentlyprovided as cultures per se or treated matters thereof, wherein saidtreated matters are selected from the group consisting of concentratedculture, dried culture, cells obtained by centrifuging the culture,products obtained by treating the cells by drying, freeze-drying,ultrasonication, mechanical friction, protein fractionation andcell-immobilization, and an enzyme preparation obtained by extractingthe cells, or a preparation obtained by treatment with enzymes,surfactant or solvent, and wherein said treated matter continues to havesaid enzyme activity.
 7. The process according to any one of claims 1,2, 3 and 5, wherein said microogranism having N-acetylneuraminic acidaldolase activity is a microorganism belonging to the genus Escherichiaor Corynebacterium.
 8. The process according to any one of claims 1, 2,4 and 6, wherein said microorganism having N-acetylneuraminic acidsynthetase activity is a microorganism belonging to a genus selectedfrom the group consisting of Escherichia, Neisseria and Streptococcus.9. The process according to any one of claims 1, 2, 3 and 5, whereinsaid microorganism capable of producing pyruvic acid is a microorganismbelonging to a genus selected from the group consisting of Escherichia,Corynebacterium and Saccharomyces.
 10. The process according to any oneof claims 1, 2, 4 and 6, wherein said microorganism capable of producingphosphoenolpyruvic acid is a microorganism belonging to a genus selectedfrom the group consisting of Escherichia, Corynebacterium andSaccharomyces.
 11. The process according to claim 7, wherein saidmicroorganism belonging to the genus Escherichia is Escherichia coli.12. The process according to claim 7, wherein said microorganismbelonging to the genus Corynebacterium is Corynebacterium ammoniagenes,Corynebacterium glutamicum or Corynebacterium acetoacidophilum.
 13. Theprocess according to claim 8, wherein said microorganism belonging tothe genus Escherichia is Escherichia coli.
 14. The process according toclaim 9, wherein said microorganism belonging to the genus Escherichiais Escherichia coli.
 15. The process according to claim 10, wherein saidmicroorganism belonging to the genus Escherichia is Escherichia coli.16. The process according to claim 9, wherein said microorganismbelonging to the genus Corynebacterium is Corynebacterium ammoniagenes,Corynebacterium glutamicum or Corynebacterium acetoacidophilum.
 17. Theprocess according to claim 10, wherein said microorganism belonging tothe genus Corynebacterium is Corynebacterium ammoniagenes,Corynebacterium glutamicum or Corynebacterium acetoacidophilum.
 18. Theprocess according to claim 3 or 5, wherein said culture of amicroorganism harboring DNA encoding N-acetylglucosamine 2-epimerase iscopresent with said cultures of a microorganism havingN-acetylneuraminic acid aldolase activity and said culture ofmicroorganism capable of producing pyruvic acid.
 19. The processaccording to claim 4 or 6, wherein said culture of a microorganismharboring DNA encoding N-acetylglucosamine 2-epimerase is copresent withsaid cultures of a microorganism having N-acetylneuraminic acidsynthetase activity and said culture of microorganism capable ofproducing phosphoenolpyruvic acid.